Methylation and Demethylation of DNA in the Higher Animal

高等动物 DNA 的甲基化和去甲基化

• 批准号: 06680613
• 负责人: TAJIMA Shoji
• 金额: $ 1.47万
• 依托单位: OSAKA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680613/
• 关键词: DNA methylation; DNA methyltransferase; DNA binding protein; Myogenesis; MyoD; MyoD1

In the project, I aimed to shed light on the regulation of the DNA methylation of chromosomal DNA in vertebrate. For this purpose, I engaged in the following experiments and got the results as follows.1. I have isolated DNA methyltransferase (MTase) cDNAs from chiken (avian) and Xenopus laevis (amphibian) and determined the nucleotide sequences. Both cDNAs were expressed in cultivated cells and their properties were analyzes. Overexpressed mouse MTase showed higher de novo methylation activity than chicken MTase.2. I have identified a novel DNA binding protein, MMBP-3, that specifically recognizes the methylated form of c-Myc binding motif. The DNA binding activity of the MMBP-3 is high in the proliferating cells and decreases when the cell proliferation is arrested.3. Overexpression of mouse MTase in myoblasts accelerates the myotube formation. This is caused by direct induction of MyoD gene transcription under proliferating conditions. The expression of MyoD gene positively correlates with the methylation of specific CpG sequences near transcriptional start site.4. I have isolated a novel cDNA named AZ1, of which expression is induced with demethylating reagent. The AZ1 protein is localized to the pre-acrosome of spermatocytes.

在这个项目中，我的目标是阐明脊椎动物染色体DNA甲基化的调控。为此，我进行了以下实验，得到了如下结果.我已经从鸡（鸟类）和非洲爪蟾（两栖动物）中分离出DNA甲基转移酶（MTase）cDNA，并测定了核苷酸序列。在培养的细胞中表达两种cDNA并分析其性质。过表达的小鼠MTase表现出比鸡MTase更高的从头甲基化活性.我发现了一种新型DNA结合蛋白MMBP-3，它特异性识别c-Myc结合基序的甲基化形式。MMBP-3的DNA结合活性在增殖期细胞中较高，当细胞增殖停止时降低.小鼠MTase在成肌细胞中的过表达加速了肌管的形成。这是由增殖条件下MyoD基因转录的直接诱导引起的。MyoD基因的表达与转录起始位点附近特定CpG序列的甲基化正相关.我已经分离了一个新的cDNA命名为AZ 1，它的表达是用去甲基化试剂诱导的。AZ 1蛋白定位于精母细胞顶体前。

项目成果

Takao Maruyama: "A Single point mutation in the splice donor sile of the low deusity lipoprotein receptor gene produces intron read-through,exon-skipped and cryptic site utilized transcripts" European Journal of Biochemistry. 232. 700-705 (1995)

Takao Maruyama：“低密度脂蛋白受体基因的剪接供体区域中的单点突变产生内含子通读、外显子跳过和神秘位点利用的转录本”《欧洲生物化学杂志》。

J.D.MILLER et al.: "The beta-subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha-subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane." Journal of Cell Biology. 128. 273-282 (1995)

J.D.MILLER 等人：“信号识别颗粒受体的 β 亚基是一种跨膜 GTP 酶，它将 α 亚基（一种外周膜 GTP 酶）锚定到内质网膜上。”

Shoji TAJIMA et al.: "Isolation and expression of a chicken DNA methyltransferase cDNA." Journal of Biochemistry. 117. 1050-1057 (1995)

Shoji TAJIMA 等人：“鸡 DNA 甲基转移酶 cDNA 的分离和表达。”

Takao MARUYAMA et al.: "A single point mutation in the splice donor site of the low density lipoprotein receptor gene produces intron read-through, exonskipped and cryptic site utilized transcripts." European Journal of Biochemistry. 232. 700-705 (1995)

Takao MARUYAMA 等人：“低密度脂蛋白受体基因剪接供体位点的单点突变产生内含子通读、外显子跳过和隐藏位点利用的转录本。”

Muroya,S.: "Selective inhibition of a step of myotube formation with wheat germ oggulutimin in a murire myoblast cell line,C2C12" Cell Structure and Function. 19. 241-252 (1994)

Muroya,S.：“在小鼠成肌细胞系 C2C12 中用小麦胚芽奥古鲁肽选择性抑制肌管形成的步骤”细胞结构和功能。